DOCSLIB.ORG

A Broadening View of Recombinational DNA Repair in Bacteria

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
A Broadening View of Recombinational DNA Repair in Bacteria REVIEW A broadening view of recombinational DNA repair in bacteria Michael M. Cox* Department of Biochemistry, University of Wisconsin-Madison, 420 Henry Mall, Madison, WI 53706, USA Recombinational DNA repair is both the most complex and least understood of DNA repair pathways. In bacterial cells grown under normal laboratory conditions (without a DNA damaging treatment other than an aerobic environment), a substantial number (10–50%) of the replication forks originating at oriC encounter a DNA lesion or strand break. When this occurs, repair is mediated by an elaborate set of recombinational DNA repair pathways which encompass most of the enzymes involved in DNA metabolism. Four steps are discussed: (i) The replication fork stalls and/or collapses. (ii) Recombination enzymes are recruited to the location of the lesion, and function with nearly perfect efficiency and fidelity. (iii) Additional enzymatic systems, including the fX174-type primosome (or repair primosome), then function in the origin- independent reassembly of the replication fork. (iv) Frequent recombination associated with recombinational DNA repair leads to the formation of dimeric chromosomes, which are monomerized by the XerCD site-specific recombination system. contribution to bacterial DNA replication under Introduction normal growth conditions. A summary of the likely Recombinational DNA repair represents a cross-roads fate of a replication fork in the Escherichia coli where virtually every aspect of DNA metabolism comes chromosome can serve as an overview to organize this together. When a bacterial cell is subjected to UV discussion (Fig. 1). Once initiated at oriC, some irradiation or other DNA damaging treatment, DNA replication forks complete their task, while others replication rapidly comes to a halt. After 30–40 min, encounter either an unrepaired DNA lesion or a DNA replication is restored to its original level. Replication strand break at a lesion undergoing repair. At these restart (Khidhir et al. 1985; Echols & Goodman 1990; encounters, the replication complex halts and/or Echols & Goodman 1991) requires both recombination collapses. The resulting gap or double-strand break is and replication functions. In the meantime, a wide array processed by recombination enzymes. The branched of DNA repair processes are induced as part of the SOS DNA replication fork is re-established after a lag of system, including many that facilitate the recombina- some minutes. A replication complex which may be tional DNA repair and replication restart. Information distinctly different from that assembled at oriC, comes about what occurs during the 30–40 min required for together in an origin-independent manner, and replica- replication to recover is still limited, but available tion again proceeds unimpeded. Any DNA lesions left experimental data can provide some insight. behind are now within double-stranded DNA and can The transient abatement of DNA synthesis is almost be processed by excision repair pathways. The improper certainly not limited to environmental stress producing resolution of recombination intermediates (Holliday unusual levels of DNA damage. Instead it is a structures) producing a dimeric bacterial chromosome manifestation (albeit dramatic and readily observed) of is countered by a distinct and specialized site-specific a process that makes an important and regular recombination system. The pathways presented in Fig. 1 are not intended * Correspondence: E-mail: [email protected] to be comprehensive or unique, and the precise q Blackwell Science Limited Genes to Cells (1998) 3, 65–78 65 MM Cox primarily to address the requirements of recombina- tional DNA repair. The importance of this process is also oriC - dependent replication reflected in an extraordinary concentration of facilitating (DNA polymerase III DnaB, DnaC, DnaG) sequences (chi sites) seen throughout the E. coli genome. A concise integration requires a focus on several GAP repair DS break repair themes. First, a premature termination of replication fork movement which requires recombinational DNA DNA lesion DNA nick repair is a very common occurrence even in the absence of 1 Replication fork 1 Replication fork disassembly collapse treatments designed to elevate DNA damage. Second, the re-initiation of replication after recombinational repair requires a specialized enzymatic system and mechanisms distinct from those applied at the genomic origin. Recombination Recombination Third, the organization of the replication fork may be 2 (RecA + RecFOR) 2 (RecBCD RecA) altered after recombinational DNA repair. Fourth, homologous genetic recombination is a required step in this repair pathway, bringing with it an array of genomic and cellular consequences. resolution resolution (RuvABC + RecG) (RuvABC or RecG) The paradigm outlined in Fig. 1 can enhance our 3 understanding of the genetics and biochemistry of the Origin-independent replication proteins playing a direct or indirect role in recombina- re-start tional DNA repair. It also has the potential to illuminate Repair Primosome a surprising number of biological systems, enzymatic activities and cellular phenomena that have sometimes been difficult to place in an appropriate functional origin-independent replication 4 -Termination context. -Dimer resolution (XerCD) Figure 1 Pathways of recombinational DNA repair. The steps described in the text are outlined. The listing of proteins involved The structure of replication forks assembled at in each step is not meant to be exhaustive. Circled numbers oriC correspond to the four steps highlighted in the text. Following an oriC-dependent initiation process that relies on the activities of the DnaA and DnaC proteins, replication forks proceed bidirectionally from the E. coli mechanisms by which these processes occur are left origin (Kornberg & Baker 1992; Marians 1992). The intentionally vague. Many of the ideas in Fig. 1 can be contiguous protein complex at each fork consists of the traced to recombinational repair models presented by asymmetric DNA polymerase III holoenzyme and the West and Howard-Flanders (West et al. 1981) and Szostak DnaB helicase (Fig. 2). The DnaG primase plays an et al.(Szostaket al.1983).Manyofthesameideashavealso intermittent role in the priming of lagging strand DNA been developed in a number of recent articles, books and synthesis (Tougu & Marians 1996). Auxiliary proteins reviews (Zavitz & Marians 1991; Cox 1993; Livneh et al. (DNA topoisomerases, single-strand DNA binding 1993; Asai et al. 1994a; Friedberg et al. 1995; Sherratt et al. protein (SSB)) play important roles that do not 1995; Kuzminov 1996a; Kogoma 1997; Roca & Cox necessarily require direct physical contact with the 1997). Drawing from different perspectives, each con- replication fork complex. The result is an integrated tributes to the synthesis attempted here and should be complex that carries out DNA synthesis on both DNA consulted for more detailed discussions of individual template strands (Fig. 2). points and for some alternative views. Continuous DNA synthesis on the leading strand is Bacteria have made an extraordinary evolutionary complemented by the coordinated synthesis of Okazaki investment in recombinational DNA repair. At some fragments on the lagging strand. The DnaG primase point in the scheme of Fig. 1, almost every bacterial interacts transiently with the DnaB helicase (Tougu & protein known to play a role in DNA metabolism leaves Marians 1996) to effect routine priming of lagging its mark. Many of these proteins, particularly those with strand DNA synthesis. For replication which is initiated the designations Rec, Pri and Xer, may have evolved at oriC, the DnaB and DnaG proteins constitute a 66 Genes to Cells (1998) 3, 65–78 q Blackwell Science Limited Recombinational DNA repair in bacteria A 5' minimal (oriC-type) lagging strand primosome, both in 3' vivo and in vitro (Kornberg & Baker 1992; Marians 1992). Both of these proteins are essential in E. coli, and 3' ts mutants exhibit a rapid-stop phenotype with respect core 5' γ τ leading strand to DNA synthesis (Wechsler & Gross 1971). The DnaB complex protein is the only one of the multiple E. coli helicases DnaB core lagging strand that is absolutely required for chromosomal replication helicase (Baker et al. 1986). RNA primer Conspicuously absent from the complex at the RNA primer replication fork are five of the proteins defined as 5'3' components of a larger E. coli primosome, often primase referred to as the fX174-type primosome. These B include the PriA, PriB, PriC, DnaC and DnaT proteins, originally discovered during in vitro studies of the replication of the bacteriophage fX174 genome (Arai DnaB helicase & Kornberg 1981; Zavitz & Marians 1991). This more elaborate primosome is required for initiation of DnaG replication and lagging strand synthesis in bacteriophage primase fX174 and also for plasmids with ColE1 origins (Zavitz new & Marians 1991). However, a fX174-type primosome RNA primer is not required for replication originating at oriC, either in vivo or in vitro. This opens the intriguing question of the cellular function of a fX174-type primosome, which presumably did not evolve to serve the needs of β primase bacteriophages or plasmids. C The PriA protein plays a central role in the assembly of the fX174-type primosome, and also exhibits DNA- dependent ATPase and DNA helicase activities in vitro (Wickner & Hurwitz 1975; Shlomai & Kornberg 1980). The issue of primosome function has
Load more

Recommended publications
  • P1 Bacteriophage and Tol System Mutants
    P1 BACTERIOPHAGE AND TOL SYSTEM MUTANTS Cari L. Smerk A Thesis Submitted to the Graduate College of Bowling Green State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE August 2007 Committee: Ray A. Larsen, Advisor Tami C. Steveson Paul A. Moore Lee A. Meserve ii ABSTRACT Dr. Ray A. Larsen, Advisor The integrity of the outer membrane of Gram negative bacteria is dependent upon proteins of the Tol system, which transduce cytoplasmic-membrane derived energy to as yet unidentified outer membrane targets (Vianney et al., 1996). Mutations affecting the Tol system of Escherichia coli render the cells resistant to a bacteriophage called P1 by blocking the phage maturation process in some way. This does not involve outer membrane interactions, as a mutant in the energy transucer (TolA) retained wild type levels of phage sensitivity. Conversely, mutations affecting the energy harvesting complex component, TolQ, were resistant to lysis by bacteriophage P1. Further characterization of specific Tol system mutants suggested that phage maturation was not coupled to energy transduction, nor to infection of the cells by the phage. Quantification of the number of phage produced by strains lacking this protein also suggests that the maturation of P1 phage requires conditions influenced by TolQ. This study aims to identify the role that the TolQ protein plays in the phage maturation process. Strains of cells were inoculated with bacteriophage P1 and the resulting production by the phage of viable progeny were determined using one step growth curves (Ellis and Delbruck, 1938). Strains that were lacking the TolQ protein rendered P1 unable to produce the characteristic burst of progeny phage after a single generation of phage.
    [Show full text]
  • Scalable Recombinase-Based Gene Expression Cascades
    bioRxiv preprint doi: https://doi.org/10.1101/2020.06.20.161430; this version posted June 20, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. Title: Scalable recombinase-based gene expression cascades One Sentence Summary: Recombinase-based gene circuits enable scalable and sequential gene modulation. Authors: Tackhoon Kim1,2, Benjamin Weinberg3, Wilson Wong3, Timothy K. Lu1,* Affiliations: 1 Research Lab of Electronics, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA. 2 Chemical Kinomics Research Center, Korea Institute of Science and Technology, 5 Hwarangro 14-gil, Seongbuk-gu, Seoul 02792, Republic of Korea 3 Department of Biomedical Engineering and Biological Design Center, Boston University, Boston, Massachusetts, USA. *Correspondence to: [email protected] (T.K.L) Abstract: Temporal modulation of multiple genes underlies sophisticated biological phenomena. However, there are few scalable and generalizable gene circuit architectures for the programming of sequential genetic perturbations. We describe a modular recombinase-based gene circuit architecture, comprising tandem gene perturbation cassettes (GPCs), that enables the sequential expression of multiple genes in a defined temporal order by alternating treatment with just two orthogonal ligands. We used tandem GPCs to sequentially express single-guide RNAs to encode transcriptional cascades and trigger the sequential accumulation of mutations. We built an all-in- one gene circuit that sequentially edits genomic loci, synchronizes cells at a specific stage within bioRxiv preprint doi: https://doi.org/10.1101/2020.06.20.161430; this version posted June 20, 2020.
    [Show full text]
  • Capture of a Recombination Activating Sequence from Mammalian Cells
    Gene Therapy (1999) 6, 1819–1825 1999 Stockton Press All rights reserved 0969-7128/99 $15.00 http://www.stockton-press.co.uk/gt Capture of a recombination activating sequence from mammalian cells P Olson and R Dornburg The Dorrance H Hamilton Laboratories, Center for Human Virology, Division of Infectious Diseases, Jefferson Medical College, Thomas Jefferson University, Jefferson Alumni Hall, 1020 Locust Street, Rm 329, Philadelphia, PA 19107, USA We have developed a genetic trap for identifying revealed a putative recombination signal (CCCACCC). sequences that promote homologous DNA recombination. When this heptamer or an abbreviated form (CCCACC) The trap employs a retroviral vector that normally disables were reinserted into the vector, they stimulated vector itself after one round of replication. Insertion of defined repair and other DNA rearrangements. Mutant forms of DNA sequences into the vector induced the repair of a 300 these oligomers (eg CCCAACC or CCWACWS) did not. base pair deletion, which restored its ability to replicate. Our data suggest that the recombination events occurred Tests of random sequence libraries made in the vector within 48 h after transfection. Keywords: recombination; retroviral vector; vector stability; gene conversion; gene therapy Introduction scripts are still made from the intact left LTR, but reverse transcription copies the deletion to both LTRs, disabling Recognition of cis-acting DNA signals occupies a central the daughter provirus.15–17 We found that the SIN vectors role in both site-specific and general recombination path- that could escape this programmed disablement did so 1–6 ways. Signals in site-specific pathways define the by recombinationally repairing the U3-deleted LTR.
    [Show full text]
  • Recombination Hotspot ANDREW F
    Proc. Natd. Acad. Sci. USA Vol. 89, pp. 5226-5230, June 1992 Biochemistry RecBCD enzyme is altered upon cutting DNA at a Chi recombination hotspot ANDREW F. TAYLOR AND GERALD R. SMITH Fred Hutchinson Cancer Research Center, 1124 Columbia Street, Seattle, WA 98104 Communicated by Hamilton 0. Smith, March 16, 1992 ABSTRACT During its unidirectional unwinding of DNA, to ensure even numbers of exchanges in conjugal and trans- RecBCD enzyme cuts one DNA strand near a properly oriented ductional crosses (13). At each end ofthe donor fragment one Chi site, a hotspot of homologous genetic recombination in RecBCD molecule is proposed to enter and promote just one Escherichia cohl. We report here that individual DNA molecules exchange. Such a mechanism would ensure exactly two containing two properly oriented Chi sites were cut with about exchanges and, hence, viability. 40% efficiency at one or the other Chi site but not detectably By what mechanism might RecBCD promote just one at both Chi sites. Furthermore, initial incubation of RecBCD exchange near each end ofthe donor fragment? This problem with Chi-containing DNA reduced its ability both to unwind is compounded by the high density of Chi sites in E. coli DNA and to cut at Chi sites on subsequently added DNA DNA: Chi occurs, on the average, once every 5 kilobases (kb) molecules much more than did initial incubation with Chi-free (18). A conjugational donor fragment one-quarter of the DNA; the nuclease activity was less severely affected. These chromosome long would contain about 250 Chi sites. Coor- results imply that RecBCD loses its Chi-cutting activity upon dination among the multitude of potential exchanges at these cutting at a single Chi site and provide a mechanism for sites seems difficult.
    [Show full text]
  • Identification and Analysis of Recombineering Functions from Gram-Negative and Gram-Positive Bacteria and Their Phages
    Identification and analysis of recombineering functions from Gram-negative and Gram-positive bacteria and their phages Simanti Datta, Nina Costantino, Xiaomei Zhou, and Donald L. Court† Gene Regulation and Chromosome Biology Laboratory, Center for Cancer Research, National Cancer Institute at Frederick, Frederick, MD 21702 Edited by Sankar Adhya, National Institutes of Health, Bethesda, MD, and approved December 12, 2007 (received for review September 25, 2007) We report the identification and functional analysis of nine genes but linear DNA recombination studies with RecET have used ␭ from Gram-positive and Gram-negative bacteria and their phages Gam to inhibit nucleases (4). that are similar to lambda (␭) bet or Escherichia coli recT. Beta and For recombineering, either a dsDNA PCR product (1, 4, RecT are single-strand DNA annealing proteins, referred to here as 15–17) or an oligonucleotide (oligo) (18–21) carrying short recombinases. Each of the nine other genes when expressed in E. (Ϸ50-bp) segments homologous to the target sequences can be coli carries out oligonucleotide-mediated recombination. To our used. These linear DNA substrates are precisely recombined in knowledge, this is the first study showing single-strand recombi- vivo by the phage proteins to target DNA on any replicon. When nase activity from diverse bacteria. Similar to bet and recT, most of linear dsDNA is used for recombination, both the exonuclease these other recombinases were found to be associated with pu- and single-strand annealing protein are required. For optimal tative exonuclease genes. Beta and RecT in conjunction with their recombination, RecBCD should be inactivated, either by muta- cognate exonucleases carry out recombination of linear double- tion or by ␭ Gam (1, 15, 22).
    [Show full text]
  • Gerry Smith Publications 1971-1999
    Gerry Smith Publications 1971-1999 Hong J-S, Smith GR, Ames BN. Adenosine 3':5'-cyclic monophosphate concentration in the bacterial host regulates the viral decision between lysogeny and lysis. Proc Natl Acad Sci USA 1971; 68: 2258-2262. Smith GR. Specialized transduction of the Salmonella hut operons by coliphage : Deletion analysis of the hut operons employing phut. Virology 1971; 45: 208- 223. Smith GR, Halpern YS, Magasanik B. Genetic and metabolic control of enzymes responsible for histidine degradation in Salmonella typhimurium. J Biol Chem 1971; 246: 3320-3329. Smith GR, Magasanik B. Nature and self-regulated synthesis of the repressor of the hut operons in Salmonella typhimurium. Proc Natl Acad Sci USA 1971; 68: 1493-1497. Smith GR, Magasanik B. The two operons of the histidine utilization system in Salmonella typhimurium. J Biol Chem 1971; 246: 3330-3341. Singer CE, Smith GR. Histidine regulation in Salmonella typhimurium: XIII. Nucleotide sequence of histidine transfer ribonucleic acid. J Biol Chem 1972; 247: 2989-3000. Singer CE, Smith GR, Cortese R, Ames BN. Mutant tRNAHis ineffective in repression and lacking two pseudouridine modifications. Nature New Biol 1972; 238: 72-74. Smith GR, Tong B. Construction of 80dhis carrying Salmonella typhimurium histidine operon mutations. J Bacteriol 1974; 120: 1223-1226. Smith GR. Deletion mutations of the immunity region of coliphage . Virology 1975; 64: 544-552. Smith GR, Hedgpeth J. Oligo (A) not coded by DNA generating 3'-terminal heterogeneity in a phage RNA. J Biol Chem 1975; 250: 4818-4821. Smith GR, Eisen H, Reichardt L, Hedgpeth J. Deletions of lambda phage locating a prm mutation within the rightward operator.
    [Show full text]
  • Recbcd Is Required to Complete Chromosomal Replication: Implications for Double-Strand Break Frequencies and Repair Mechanisms
    DNA Repair 32 (2015) 86–95 Contents lists available at ScienceDirect DNA Repair journal homepage: www.elsevier.com/locate/dnarepair RecBCD is required to complete chromosomal replication: Implications for double-strand break frequencies and repair mechanisms Justin Courcelle ∗, Brian M. Wendel, Dena D. Livingstone, Charmain T. Courcelle Department of Biology, Portland State University, Portland, OR 97201, United States article info abstract Article history: Several aspects of the mechanism of homologous double-strand break repair remain unclear. Although Available online 2 May 2015 intensive efforts have focused on how recombination reactions initiate, far less is known about the molec- ular events that follow. Based upon biochemical studies, current models propose that RecBCD processes Keywords: double-strand ends and loads RecA to initiate recombinational repair. However, recent studies have Double-strand break repair shown that RecBCD plays a critical role in completing replication events on the chromosome through a Homologous recombination mechanism that does not involve RecA or recombination. Here, we examine several studies, both early Replication completion and recent, that suggest RecBCD also operates late in the recombination process – after initiation, strand RecBCD invasion, and crossover resolution have occurred. Similar to its role in completing replication, we propose a model in which RecBCD is required to resect and resolve the DNA synthesis associated with homologous recombination at the point where the missing sequences on the broken molecule have been restored. We explain how the impaired ability to complete chromosome replication in recBC and recD mutants is likely to account for the loss of viability and genome instability in these mutants, and conclude that spontaneous double-strand breaks and replication fork collapse occur far less frequently than previously speculated.
    [Show full text]
  • Cre/Lox-Mediated Chromosomal Integration of Biosynthetic Gene Clusters for Heterologous Expression in Aspergillus Nidulans
    bioRxiv preprint doi: https://doi.org/10.1101/2021.08.20.457072; this version posted August 20, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. Cre/lox-mediated chromosomal integration of biosynthetic gene clusters for heterologous expression in Aspergillus nidulans Indra Roux1, Yit-Heng Chooi1 1 School of Molecular Sciences, University of Western Australia, Perth, WA 6009, Australia. Correspondence to: [email protected] Abstract Building strains for stable long-term heterologous expression of large biosynthetic pathways in filamentous fungi is limited by the low transformation efficiency or genetic stability of current methods. Here, we developed a system for targeted chromosomal integration of large biosynthetic gene clusters in Aspergillus nidulans based on site-specific recombinase mediated cassette exchange. We built A. nidulans strains harbouring a chromosomal landing pad for Cre/lox-mediated recombination and demonstrated efficient targeted integration of a 21.5 kb heterologous region in a single step. We further evaluated the integration at two loci by analysing the expression of a fluorescent reporter and the production of a heterologous polyketide. We compared chromosomal expression at those landing loci to episomal AMA1- based expression, which also shed light on uncharacterised aspects of episomal expression in filamentous fungi.
    [Show full text]
  • Chi-Dependent Intramolecular Recombination in Escherichia Coli
    Copyright © 1998 by the Genetics Society of America Chi-Dependent Intramolecular Recombination in Escherichia coli Rachel Friedman-Ohana, Iris Karunker and Amikam Cohen Department of Molecular Biology, The Hebrew University —Hadassah Medical School, Jerusalem, Israel 91010 Manuscript received June 13, 1997 Accepted for publication October 17, 1997 ABSTRACT Homologous recombination in Escherichia coli is enhanced by a cis-acting octamer sequence named Chi (59-GCTGGTGG-39) that interacts with RecBCD. To gain insight into the mechanism of Chi-enhanced re- combination, we recruited an experimental system that permits physical monitoring of intramolecular re- combination by linear substrates released by in vivo restriction from infecting chimera phage. Recombina- tion of the released substrates depended on recA, recBCD and cis-acting Chi octamers. Recombination proficiency was lowered by a xonA mutation and by mutations that inactivated the RuvABC and RecG reso- lution enzymes. Activity of Chi sites was influenced by their locations and by the number of Chi octamers at each site. A single Chi site stimulated recombination, but a combination of Chi sites on the two homologs was synergistic. These data suggest a role for Chi at both ends of the linear substrate. Chi was lost in all re- combinational exchanges stimulated by a single Chi site. Exchanges in substrates with Chi sites on both ho- mologs occurred in the interval between the sites as well as in the flanking interval. These observations sug- gest that the generation of circular products by intramolecular recombination involves Chi-dependent processing of one end by RecBCD and pairing of the processed end with its duplex homolog.
    [Show full text]
  • Coupling Recombinase-Mediated Cassette Exchange with Somatic Hypermutation for Antibody Affinity Maturation in CHO Cells
    ARTICLE Coupling Recombinase-Mediated Cassette Exchange With Somatic Hypermutation for Antibody Affinity Maturation in CHO Cells Chuan Chen,1,2 Nan Li,1,2 Yun Zhao,1 Haiying Hang1 1 Key Laboratory for Protein and Peptide Pharmaceuticals, National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China; telephone: þ86-10-64888473; fax: þ86-10-64888473; e-mail: [email protected] 2 University of Chinese Academy of Sciences, Beijing, China Introduction ABSTRACT: Heterologous expression of activation-induced cytidine deaminase (AID) can induce somatic hypermutation Technologies such as phage (de Bruin et al., 1999; Huse et al., 1992; (SHM) for genes of interest in various cells, and several research Smith, 1985; Winter et al., 1994), yeast (Boder and Wittrup, 1997; groups (including ours) have successfully improved antibody affinity in mammalian or chicken cells using AID-induced SHM. These Feldhaus et al., 2003), and bacterial displays (Francisco and affinity maturation systems are time-consuming and inefficient. In Georgiou, 1994; Mazor et al., 2009; Qiu et al., 2010) have been used this study, we developed an antibody affinity maturation platform in for antibody affinity maturation in vitro. In recent years, Chinese hamster ovary (CHO) cells by coupling recombinase- mammalian cell surface display has also been developed (Akamatsu mediated cassette exchange (RMCE) with SHM. Stable CHO cell et al., 2007; Higuchi et al., 1997; Ho et al., 2006; Wolkowicz et al., clones containing a single copy puromycin resistance gene (PuroR) expression cassette flanked by recombination target sequences (FRT 2005); it has advantages in protein folding, post-translational and loxP) being able to highly express a gene of interest placed in the modification, and code usage (Ho et al., 2006).
    [Show full text]
  • Recbcd Enzyme “Chi Recognition” Mutants Recognize Chi Recombination Hotspots in the Right DNA Context
    | INVESTIGATION RecBCD Enzyme “Chi Recognition” Mutants Recognize Chi Recombination Hotspots in the Right DNA Context Susan K. Amundsen, Jake W. Sharp,1 and Gerald R. Smith2 Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, Washington 98109 ORCID ID: 0000-0002-8747-1352 (G.R.S.) ABSTRACT RecBCD enzyme is a complex, three-subunit protein machine essential for the major pathway of DNA double-strand break repair and homologous recombination in Escherichia coli. Upon encountering a Chi recombination-hotspot during DNA unwinding, RecBCD nicks DNA to produce a single-stranded DNA end onto which it loads RecA protein. Conformational changes that regulate RecBCD’s helicase and nuclease activities are induced upon its interaction with Chi, defined historically as 59 GCTGGTGG 39. Chi is thought to be recognized as single-stranded DNA passing through a tunnel in RecC. To define the Chi recognition-domain in RecC and thus the mechanism of the RecBCD-Chi interaction, we altered by random mutagenesis eight RecC amino acids lining the tunnel. We screened for loss of Chi activity with Chi at one site in bacteriophage l. The 25 recC mutants analyzed thoroughly had undetectable or strongly reduced Chi-hotspot activity with previously reported Chi sites. Remarkably, most of these mutants had readily detectable, and some nearly wild-type, activity with Chi at newly generated Chi sites. Like wild-type RecBCD, these mutants had Chi activity that responded dramatically (up to fivefold, equivalent to Chi’s hotspot activity) to nucleotide changes flanking 59 GCTGGTGG 39. Thus, these and previously published RecC mutants thought to be Chi-recognition mutants are actually Chi context-dependence mutants.
    [Show full text]
  • Efficient Genome Engineering by Targeted Homologous
    ARTICLE Received 14 Oct 2013 | Accepted 2 Dec 2013 | Published 13 Jan 2014 | Updated 20 Feb 2015 DOI: 10.1038/ncomms4045 Efficient genome engineering by targeted homologous recombination in mouse embryos using transcription activator-like effector nucleases Daniel Sommer1, Annika E. Peters1, Tristan Wirtz1,w, Maren Mai1, Justus Ackermann2, Yasser Thabet1, Ju¨rgen Schmidt3, Heike Weighardt4, F. Thomas Wunderlich2, Joachim Degen5, Joachim L. Schultze1 & Marc Beyer1 Generation of mouse models by introducing transgenes using homologous recombination is critical for understanding fundamental biology and pathology of human diseases. Here we investigate whether artificial transcription activator-like effector nucleases (TALENs)— powerful tools that induce DNA double-strand breaks at specific genomic locations—can be combined with a targeting vector to induce homologous recombination for the introduction of a transgene in embryonic stem cells and fertilized murine oocytes. We describe the gen- eration of a conditional mouse model using TALENs, which introduce double-strand breaks at the genomic locus of the special AT-rich sequence-binding protein-1 in combination with a large 14.4 kb targeting template vector. We report successful germline transmission of this allele and demonstrate its recombination in primary cells in the presence of Cre-recombinase. These results suggest that TALEN-assisted induction of DNA double-strand breaks can facilitate homologous recombination of complex targeting constructs directly in oocytes. 1 Genomics and Immunoregulation, LIMES Institute, University of Bonn, Carl-Troll-Strasse 31, D-53115 Bonn, Germany. 2 Max Planck Institute for Neurological Research and Institute for Genetics, University of Cologne, Gleuelerstrasse 50, D-50931 Cologne, Germany. 3 Department of Experimental Therapy, University Hospital Bonn, Sigmund-Freud-Strasse 25, D-53105 Bonn, Germany.
    [Show full text]